The mechanisms initiating developmental and behavioral changes in the brain during sexual maturity remain to be elucidated. We hypothesize that hormonal factors involved in determination of sex and sexual maturity also direct recruitment of neurons into behavioral circuits. The Drosophila dopamine (DA) neurons within the stress response circuitry can serve as a model in which to elucidate these factors. As for mammals, DA is integral to the stress response pathway, DA modulates analogous behaviors, and the DA biosynthetic pathways and reuptake mechanisms are highly conserved. If unique subsets of DA neurons are recruited into the stress response circuitry in the brains of sexually immature and mature male and female Drosophila, we predict differences in DA-modulated behaviors after exposure to stress. The DA neuronal population is stereospecific and limited in number, so one can map individual DA neurons unique to each circuit. The first aim tests whether the stress response circuitry is composed of unique subsets of DA neurons in sexually immature and mature male and female Drosophila. We have identified brain regions critical for the stress response using mutant strains with distinct anatomical brain defects and will identify the individual DA neurons affected by each mutation by immunohistochemical comparisons of their neuronal DA patterns with wild type. DA levels will be decreased in those neurons by targeted knockdown of DA synthesis using transgenic and genetic tools. Deviations from the normal response in sexually immature and mature male and females will identify the individual DA neurons unique to the stress response circuitry in the different populations. We will then test whether recruitment of DA neurons into the stress circuitry is influenced by factors that regulate sexual differentiation and sexual maturity. While the population of DA neurons is not sexually dimorphic, DA neurons recruited into the response circuits differ in males and females. Feminization of subsets of neurons in an otherwise male fly should alter the stress response if neurons critical for the response have been appropriately targeted. The actions of gonadotropic hormones affect the stress response as well as neuronal differentiation and reorganization. The impact of the hormonal environment surrounding the DA neurons can be assessed using pharmacological tools. The experiments proposed in this application will identify the specific DA neurons recruited into the stress response circuitry that occur during sexual differentiation and sexual maturity, and initiate identification of hormonal mediators affecting recruitment of individual neurons. This will provide a unique and innovative approach to modeling the development and organization of the stress circuitry in mammals.